The endothelial cell lining constitutes the interface between vascular tissue and circulating blood, and plays an important role in regulation of molecular exchange between blood and perivascular tissues. Although endothelial cells are positioned optimally to interact with circulating clotting factors, very little is known about these interactions. Recently, we have shown that factor VIIa (FVIIa) binds specifically to endothelial cell protein C receptor (EPCR) on endothelial cells. Both FVII and FVIIa bind to EPCR in a true-ligand fashion and with a similar affinity as that of protein C and activated protein C (APC) to EPCR. At present the physiological significance and importance of FVII/FVIIa interaction with EPCR are unknown. Our recent studies showed that FVIIa binding to EPCR resulted in its endocytosis. These studies also indicate that EPCR-mediated endocytosis and recycling may facilitate the transport of FVIIa from the luminal to abluminal surface. Our latest studies show that FVIIa binding to EPCR on endothelial cells activates protease activated receptor-1 (PAR1) and provides endothelial barrier protection. Studies in cell model systems have suggested that therapeutic concentrations of FVIIa may down-regulate the protein C anticoagulant pathway by displacing protein C from EPCR on the endothelium. All of these findings are novel and cumulatively indicate that FVIIa interaction with EPCR may play an important role in pathophysiology. Based on these findings, we hypothesize that FVIIa interaction with EPCR on the endothelium modulates FVIIa transport, provides protection against vascular leakage, and down-regulates the protein C/APC anticoagulant pathway in therapeutical conditions. Three specific aims that test these hypotheses are, (1) investigate the role of EPCR in FVIIa transcytosis and clearance from bloodstream, (2) elucidate EPCR-FVIIa-mediated barrier protective mechanism(s) in endothelial cells and define the role of FVIIa-EPCR signaling in maintaining vascular barrier integrity in vivo, and (3) test the hypothesis that pharmacological concentrations of rFVIIa down-regulate the protein C/APC-mediated anticoagulant pathway and thereby potentiate the hemostatic effect of rFVIIa in the treatment of bleeding disorders. For these studies, we will use both cell and animal model systems. We will employ well characterized EPCR deficient and EPCR overexpressing mice and antibodies specific to mouse EPCR in order to investigate the role of EPCR in FVIIa transport and mediating FVIIa-induced cellular effects. FVIIa interaction with the endothelium and its transport will be probed by immunohistochemistry, transmission electron microscopy and measuring FVII/FVIIa activity and antigen in tissues. Confocal and fluorescence resonance energy transfer (FRET) microscopy will be used to investigate the physical interaction of FVIIa, EPCR and PAR1. The proposed studies will reveal novel functions of EPCR and FVIIa and revise our current understanding on the role of FVIIa and EPCR in hemostasis and inflammation. The proposed studies will have major and important implications for future treatment strategies of bleeding disorders and sepsis. PUBLIC HEALTH RELEVANCE: The proposed studies will provide valuable and novel information on how interaction of clotting factor VIIa with endothelial cell protein C receptor (EPCR) modulates hemostasis and inflammation. Data obtained from the proposed studies will provide new understanding to our existing knowledge on how recombinant factor VIIa functions as an effective hemostatic agent in treating hemophiliacs with inhibitors, and how prophylactic use of FVIIa can reduce joint bleeds and slow joint destruction in these patients even after cessation of treatment. Overall, the proposed studies will provide critical knowledge to improve efficacy of treatment of bleeding disorders and reduce treatment costs, and also open novel therapeutic opportunities for rFVIIa in treating inflammatory diseases.